Modification of a glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.
1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4)-glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with water-soluble carbodiimides was studied. 2. Gluc M1 was inactivated most effectively by 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide (CMC) at pH 4.5. 3. Inactivation of Gluc M1 with [14C]CMC proceeded with the incorporation of about 12 CMC moieties. From the results of amino acid analysis, titration of SH group with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of Gluc M1. 4. The CD spectrum of CMC-modified Gluc M1 (residual activity, ca. 9.8%) suggested that the gross conformation of the native enzyme was retained. 5. In the presence of maltose, when Gluc M1 was incubated with [14C]CMC, ca. 10 CMC moieties were incorporated with a simultaneous decrease in enzymatic activity (30%). The Gluc M1 modified in the presence of maltose was remodified with CMC after elimination of maltose. The CMC-modified Gluc M1 was inactivated completely with the incorporation of ca. 4 CMC moieties. 6. The logarithm of the half-life of the inactivation of Gluc M1 by CMC was a linear function of log [CMC] indicating that one carboxyl group among the modified ones was crucial for inactivation of Gluc M1. 7. The protection by maltose of Gluc M1 from inactivation and the increase in K1 values for maltose of CMC-modified Gluc M1's suggested that a crucial carboxyl group(s) was located near or on subsites 2 and 3.
D001230:Aspergillus / Q000201:enzymology*; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology*; D002234:Carbodiimides / Q000494:pharmacology*; D002942:Circular Dichroism; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors*; D005959:Glucosidases / Q000037:antagonists & inhibitors*; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000494:pharmacology; D013329:Structure-Activity Relationship
D002234:Carbodiimides; D003028:CME-Carbodiimide; D008320:Maltose; D005959:Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase